Enhanced protein thermostability and temperature resistance

ABSTRACT

Small heat shock proteins, e.g.,  Pyrococcus fuiosus  (Pfu-sHSP and/or Pfu-tsHSP), confer thermotolerance on cellular cultures and on proteins in cellular extracts during prolonged incubation at elevated temperature, demonstrating the ability to protect cellular proteins and maintain cellular viability under heat stress conditions. Such heat shock proteins are effective to combat enzymatic aggregation and intracellular precipitation during heat stress, and thereby enable enhancement of the utility and stability of enzymes in various applications, e.g., Taq polymerase in PCR applications, digestive enzymes in microbial degradative applications, etc.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of co-pending U.S.patent application Ser. No. 09/835,909 filed on Apr. 16, 2001 in thenames of Frank T. Robb and Pongpan Laksanalamai for “ENHANCE PROTEINTHERMOSTABILITY AND TEMPERATURE RESISTANCE” which claims priority toU.S. Provisional Patent Application No. 60/60/197,274 filed on Apr. 14,2000.

GOVERNMENT RIGHTS IN INVENTION

[0002] Work relating to the present invention was performed during theperformance of Grant No. 98-0935. The United States Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention generally relates to heat shock proteinsfrom Pyrococcus furiosus, to a method of protecting and extending thedurability of a recombinant DNA polymerase, and to a PCR kit.

[0005] 2. Description of the Related Art

[0006] All organisms respond to elevated temperature by specificallyinducing the expression of a set of new proteins, termed “heat shockproteins” or “HSPs.” Although this response has been known for overthirty years, the specific role of individual HSPs in the overallresponse is still largely unknown. The HSPs to which functionalcharacter has been attributed have been characterized as molecularchaperones that enable protein folding, preventing denaturation of otherproteins, or mediating proteolysis. This role, however, has only beendemonstrated for a few of the many known HSPs and the function of otherHSPs remains unknown. Moreover, it is not known which of the HSPs areessentials for the overall shock response except in the cases describedbelow.

[0007] All organisms have a basal level of thermotolerance that is anorganism-specific temperature threshold above, which the organisms die.Basal levels of thermotolerance are probably determined by a variety offactors, including, for example, membrane composition and the innatethermal stability of enzymes involved in normal cellular processes. Anadditional level of thermotolerance can be acquired by exposure of anorganism to sublethal processes. Such “acquired thermotolerance” isbelieved to result from the production of HSPs in response to thesublethal high temperature exposure.

[0008] HSPs have been categorized by size and DNA sequence into familiesthat are evolutionarily conserved. These families include the HSP 100,HSP 90, HSP 70, the HSP 60 and a variable class of low molecular weightproteins that range from 12-42 kDa. The HSPs found in this class of lowmolecular weight proteins are referred to as small heat shock proteinsor sHSPs. In animals, this class of low molecular weight proteins rangesfrom 20-25 kDA. In the plant kingdom, the corresponding range is 14-20kDa.

[0009] All of the low molecular weight HSPs are distinguished byconserved carboxy termini that are highly homologous to theαB-crystallin structural protein of the eye lens. αB-crystallin isitself capable of acting as a molecular chaperone, and all sHSPs havebeen demonstrated to exhibit chaperone activities in in vitroexperiments. Their role in cells has not yet been demonstrated.

[0010] While it might be assumed that the HSPs play a role inthermotolerance because of the correlation of their abundant synthesiswith exposure to increased temperature, earlier work with yeast hadsuggested that they are unimportant for the development ofthermotolerance, as elimination of a single yeast sHSP had no effect onthermotolerance. In addition, in Drosophilia cells, the use of antisensetechnology caused a specific decrease in the synthesis of the sHSP 26protein, but such decrease had no effect on thermotolerance.

[0011] In addition to being induced by temperature stress, many HSPs,including those in the sHSP class, can be induced by other stresses suchas exposure to arsenite, ethanol, heavy metals, amino acid analogs (Lee,Y. R., et al., Plant Physio. 110:241-48 (1996); and Nover, L., (ed.)Heat Shock Response, CRC Press (1990)) and water stress (Almoguera, C.,et al., The Plant Journal 4(6):947-58 (1993). In addition, increasingnumbers of HSPs and HSP homologs are found to be regulated indevelopmental and tissue-specific ways (see, e.g., Almoguera, C. and J.Jordano, Plant Molecular Biol. 19:781-92 (1992); Apuya, N. R. and J. L.Zimmerman, The Plant Cell, 4:657-65 (1992); Cordewener, J. H. G., etal., Plant Cell 1:1137-1140 (1989). Proteins with highly conservedsequences related to HSPs, HSP cognates, may be expressed innon-stressed normal cells, but are not induced by thermal stress.

[0012] The mechanisms of action for the small HSPs are not clearlyunderstood at present. There is a need for a better understanding ofsHSPs despite other recombinant archael sHSPs that have beenoverexpressed in E. coli.

[0013] The present invention embodies an advance in the field of sHSPsthat correlatively advances the understanding of the mechanism of sHSPs.

SUMMARY OF THE INVENTION

[0014] The invention relates to heat shock proteins and their methods ofuse.

[0015] In one aspect, the invention relates to a purified and isolatednucleic acid sequence encoding a heat shock protein comprising SEQ IDNO. 1 or SEQ ID NO. 5.

[0016] Another aspect of the invention relates to the protein encoded bythe nucleic acid comprising at SEQ ID NO. 1 or SEQ ID NO. 5 and tocompositions comprising at least one of the sequences.

[0017] Another aspect of the invention relates to a protein comprisingthe amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 6, and tocompositions comprising at least one of the proteins.

[0018] A still further aspect of the invention relates to a method ofprotecting and extending the durability of a recombinant DNA polymerase,comprising the steps of:

[0019] purifying a low molecular weight heat shock protein;

[0020] adding the heat shock protein to a buffer solution containing thepolymerase;

[0021] incubating the solution at extended temperature for extendedtime;

[0022] adding components necessary for PCR;

[0023] thermocycling the reaction to produce product from amplificationof genomic deoxyribonucleic acid; and

[0024] examining the product of the reaction by gel electrophoresis.

[0025] Yet another aspect of the invention relates to a method ofmaintaining proteins in solution, comprising the steps of:

[0026] adding a low molecular weight heat shock protein to the solution;

[0027] elevating the temperature of the solution; and

[0028] measuring the enzymatic activity by absorbance.

[0029] A still further aspect of the invention relates to a PCR kitcomprising the protein encoded by the nucleic acid comprising SEQ ID NO.1, SEQ ID NO. 5 or a combination of SEQ ID NOs. 1 and 5, or the proteincomprising the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 6 or acombination of SEQ ID NOs. 2 and 6, and one or more other PCR reagents.

[0030] In a further compositional aspect, the invention relates to acomposition comprising (i) a biological component and (ii) an HSP or aprecursor thereof, which is (A) exogenous to the biological component,and (B) thermostabilizingly effective for the biological component inthe composition.

[0031] The invention contemplates in various further aspects:

[0032] a method of enhancing the stability of Taq polymerase in a PCRoperation, by conducting the PCR operation in the presence of a HSP, andpreferably the HSP is Pfu-tsHSP;

[0033] a PCR kit including PCR primers, Taq polymerase,deoxyribonucleoside triphosphates and an HSP, wherein the HSP isPfu-tsHSP;

[0034] transformed cells capable of expressing Pfu-tsHSP and/or acombination of Pfu-tsHSP and Pfu-sHSP;

[0035] recombinant DNA vectors for expression of Pfu-tsHSP and/or acombination of Pfu-tsHSP and Pfu-sHSP; and

[0036] a method of stabilizing a protein solution, including a firstprotein therein, against heat-mediated agglomeration of the firstprotein in the solution, by incorporating in the solution a heat shockprotein that is non-endogenous with respect to the first protein.

[0037] Other aspects, features and embodiments of the invention will bemore fully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 is the nucleotide sequence (SEQ ID NO. 1; GenBank AccessionNo. AF 256212) and the amino acid sequence (SEQ ID NO. 2; GenBankAccession No. AF 256212) of Pyrococcus furiosus heat shock protein.

[0039]FIG. 2 demonstrates the SDS PAGE (15%) and blot analysis ofPfu-sHSP.

[0040]FIG. 3 demonstrates the SDS PAGE (15%) analysis of thermalprotection of E. coli crude extract by Pyrococcus furiosus -sHSP at 105°C.

[0041]FIG. 4 is a graph of supernatant bovine glutamate dehydrogenaseactivity and A₂₈₀ values as a function of time.

[0042]FIG. 5 demonstrates the agarose gel electrophoresis (1%) of theprotection of Taq polymerase by the Pyrococcus furiosus -sHSP.

[0043]FIG. 6 depicts an electrophoretic gel (1% agarose) showing theeffect of Pfu-sHSP on Taq polymerase concentration in PCR reaction.

[0044]FIG. 7 depicts another electrophoretic gel (1% agarose) showingthe effect of Pfu-sHSP on Taq polymerase concentration in PCR reaction.

[0045]FIG. 8 depicts yet another electrophoretic gel (1% agarose)showing the effect of Pfu-sHSP on Taq polymerase concentration in PCRreaction.

[0046]FIG. 9 is the nucleotide sequence (SEQ ID NO. 5) and the aminoacid sequence (SEQ ID NO. 6) of Pyrococcus furiosus truncated heat shockprotein.

[0047]FIG. 10 demonstrates the SDS PAGE (15%) analysis of thermalprotection of E. coli crude extract by Pyrococcus furiosus -tsHSP at105° C.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION

[0048] Definitions

[0049] As used herein, the following terms have the following meanings.

[0050] As used herein, the terms “heat shock protein” and “truncatedheat shock protein” refer to any protein whose synthesis is enhancedwhen an organism or its cells are exposed to an increased temperaturefor that species; typically a temperature increase in a range of fromabout 5 to about 15° C.

[0051] As used herein, the term “low molecular weight heat shockprotein” refers to those heat shock proteins that are between 12-42kilodaltons (kDa) in size.

[0052] As used herein, the term “sHSP 20” refers to the small heat shockproteins encoded by SEQ ID NO. 1.

[0053] As used herein, the term “Pfu-tsHSP” refers to the truncatedsmall heat shock proteins encoded by nucleotide sequence SEQ ID NO. 5.

[0054] As used herein, the term “thermotolerance” refers to the abilityof a cell to survive exposure to temperatures above its normal growthtemperature.

[0055] As used herein, the term “basal thermotolerance” refers to themaximum temperature to which an organism or cell can survive when theshift to that temperature is rapid.

[0056] As used herein, the term “acquired thermotolerance” refers to theincrease in thermotolerance that results from a prior (pre) exposure toa sublethal heat shock temperature.

[0057] As used herein, the term “transgenic cell line” or “transgenicculture” refers to a cell or culture that has stably incorporated addedDNA sequences into its genome after deliberate introduction of DNA intothat cell.

[0058] As used herein, the term “DNA molecule” refers to the polymericform of deoxyribonucleotides (adenine, guanine, thymine or cytosine) ineither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids and chromosomes.

[0059] As used herein, the term “vector” refers to a replicon, such as aplasmid, phage, cosmid or virus to which another DNA or RNA segment maybe attached so as to bring about the replication of the attachedsegment. Specialized vectors were used herein, containing variouspromoters, polyadenylation signals, genes for selection, etc.

[0060] As used herein, the term “transcriptional and translationalcontrol sequences” refer to DNA regulatory sequences, such as promoters,enhancers, polyadenylation signals, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell.

[0061] As used herein, the term “promoter sequence” is a DNA regulatoryregion capable of binding RNA polymerase in a cell and initiatingtranscription of a downstream (3′ direction) coding sequence. Eukaryoticpromoters will often, but not always, contain “TATA” boxes and “CAT”boxes. Prokaryotic promoters contain Shine-Dalgarno sequences inaddition to the −10 and −35 consensus sequences.

[0062] As used herein, the term “selection gene” refers to a gene thatenables the discrimination of cells displaying a required phenotype uponimplementation of certain conditions. For example, the growth ofbacteria in a medium containing antibiotics to select for the bacterialcells containing antibiotic resistance genes.

[0063] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutsdouble-stranded DNA at or near a specific nucleotide sequence.

[0064] As used herein, the terms “transformed” or “transfected” byexogenous or heterogeneous DNA when such DNA has been introduced insidethe cell. The transforming DNA may or may not be integrated (covalentlylinked) into the genome of the cell. In prokaryotes, yeast and mammaliancells, for example, the transforming DNA may be maintained on anepisomal element such as a plasmid. With respect to eukaryotic cells, astably transformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clones of apopulation of daughter cells containing the transforming DNA.

[0065] As used herein, the term “clone” refers to a population of cellsderived from a single cell or common ancestor by mitosis.

[0066] As used herein, the term “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

[0067] In accordance with the present invention, conventional molecularbiology, microbiology, and recombinant DNA techniques within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: A LaboratoryManual” (1982); “DNA Cloning: A Practical Approach,” Volumes I and II(D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed.,1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds.,1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins,eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986);“Immobilized Cells & Enzymes,” IRL Press (1986); “A Practical Guide toMolecular Cloning,” B. Perbal (1984).

[0068] In a further aspect of the invention, the reagents describedherein can be packaged in a kit form for carrying out PCR. As usedherein, the term “package” refers to a solid matrix or materialscustomarily utilized in such a kit system in the form of at least one ormore enclosure that is capable of holding within fixed limits at leastone or more of the reagent components for use in PCR. Such materialsinclude glass and plastic (e.g., polyethylene, polypropylene, andpolycarbonate) bottle, vials, paper, plastic, plastic-foil laminatedenvelopes and the like. Thus, for example, a package can be a glass vialused to contain the appropriate quantities of polynucleotide primer(s),genomic DNA, vectors and DNA polymerase or a combination thereof, inaddition to an appropriate amount of sHSP and/or tsHSP. An aliquot ofeach component sufficient to perform at least one complete PCR procedureis provided in each package.

[0069] Kits useful for producing a primer extension product foramplification of a specific nucleic acid sequence using a primerextension reaction methodology also typically includes, in separatecontainer within the kit, dNTPs where N is adenine, thymine, guanine andcytosine and other like agents for performing primer extensionreactions.

[0070] The reagent species of any system described herein can beprovided in solution, as a liquid dispersion or as a substantively drypowder, e.g., the primers may be provided in lyophilized form.

[0071] The advantage of using a small heat shock protein to prevent theaggregation of mixtures of proteins is clear, but using a recombinantprotein comprising a small heat shock protein or a truncated small heatshock proteins was demonstrated by the inventors hereof to extend thehalf-life of a pure enzyme in vitro. At a temperature that was higherthan the optimal temperature and T_(max) of the enzyme, the inventorshereof demonstrated that the half-life of the enzyme incubated with therecombinant protein was increased approximately four-fold compared tothe enzyme control alone.

[0072] The heat shock proteins of the invention, Pyrococcus fuiosus(Pfu-sHSP and Pfu-tsHSP), confer thermotolerance on cellular culturesand on proteins in cellular extracts during prolonged incubation atelevated temperature, demonstrating the ability to protect cellularproteins and maintain cellular viability under heat stress conditions.Such heat shock proteins are effective to combat enzymatic aggregationand intracellular precipitation during heat stress, and thereby enableenhancement of the utility and stability of enzymes in variousapplications, e.g., Taq polymerase in PCR applications, digestiveenzymes in microbial degradative applications, etc.

[0073] The cytoprotective character of the heat shock proteins of theinvention also enables the commercial exploitation of correspondinglytransformed cellular cultures for elevated temperature fermentation andmicrobial culturing operations that take advantage of improved kineticsassociated with higher temperature processing regimes. Suchcytoprotective character further facilitates clinical applications inwhich heat-mediated undesirable side effects of pyrogenic therapeuticagents, thermal treatments and environmental exposure are combated byadministration of heat shock proteins of the invention to a human oranimal patient in need thereof (or by gene therapy producing in vivoexpression of such cytoprotective proteins).

[0074] The present invention comprehends a composition comprising (i) abiological component and (ii) an HSP or a precursor thereof, which is(A) exogenous to the biological component, and (B) thermostabilizinglyeffective for the biological component in the composition.

[0075] The invention also contemplates enhancing the stability of Taqpolymerase in a PCR operation, by conducting the PCR operation in thepresence of a HSP. The PCR operation may be carried out with theprovision of a PCR kit including PCR primers, Taq polymerase,deoxyribonucleoside triphosphates and an HSP according to the presentinvention.

[0076] Transformed cells capable of expressing Pfu-sHSP and/or Pfu-tsHSPmay be readily formed and utilized to produce Pfu-sHSP and/or Pfu-tsHSPfor various applications of the invention, e.g., by use of recombinantDNA vectors for expression of Pfu-sHSP and/or Pfu-tsHSP. The resultingheat shock protein(s) then may be employed for stabilizing a proteinsolution, including a first protein therein, against heat-mediatedagglomeration of such first protein in the solution, by incorporating inthe solution the Pfu-sHSP and/or Pfu-tsHSP or other heat shock proteinthat is non-endogenous with respect to the first protein.

[0077] The small heat shock protein from Pyrococcus furiosus (Pfu-sHSP)is composed of 167 amino acid residues encoded by an ORF of 504nucleotides (GenBank Accession number AF256212) and the truncated heatshock protein from Pyrococcus furiosus (Pfu-tsHSP) is composed of 156amino acid residues encoded by a truncated ORF of 471 nucleotides(including a stop codon).

[0078] The invention is described more specifically hereinafter, inrespect of the present inventors' work involving the gene encoding thesmall heat shock protein (sHSP) and the truncated small heat shockprotein (tsHSP) from the hyperthermophile Pyrococcus furiosus, as clonedand overexpressed in E. coli. The sHSP and tsHSP genes were found to beinducible by heat shock at 105° C. In specific experiments, recombinantsHSP and/or tsHSP prevented the majority of E. coli proteins fromaggregating in vitro for up to 40 minutes at 105° C., and also preventedbovine glutamate dehydrogenase from aggregating at 56° C. Survivabilityof E. coli overexpressing the sHSP was empirically determined to beenhanced approximately 6-fold during exposure to 50° C. for 2 hourscompared with a control culture which was not expressing sHSP. Theseresults evidence the utility of heat shock proteins fromhyperthermophiles in conferring a survival advantage on mesophilicbacteria by preventing protein aggregation at supraoptimal temperatures,and implicate usages of such heat shock proteins in microbial culturing,fermentation, and numerous other bioprocessing applications.

[0079] While the ensuing description is directed illustratively to smallheat shock proteins (sHSP and tsHSP) of Pyrococcus furiosus, it is to berecognized that the utility of the present invention is not thus limitedand that a wide variety of other sHSPs may be employed in accordancewith the present invention. Examples include, without limitation, sHSPsfrom P. horikoshii and Aquifex aeolicus.

[0080] Accordingly, the features and advantages of the invention aremore fully apparent from the following illustrative examples, which arenot intended in any way to be limitingly construed, as regards theinvention hereinafter claimed.

EXAMPLE 1

[0081] Cloning, Overexpression and Purification of the RecombinantProtein

[0082] The region encoding the Pfu-sHSP gene (SEQ ID NO. 1; GenBankAccession No. AF 256212) (FIG. 1) was amplified from Pryrococcusfuriosus genomic DNA by PCR using two primers: Pfu-shspN with an NcoIsite (underlined) (5′GCCATGGTGAGGAGAATAAGAAGATGG) (SEQ ID NO. 3) andPfu-shspC with an XhoI site (underlined)(5′ACTCGAGCTATTCAACTTTAACTTCGAATCCTTC) (SEQ ID NO. 4).

[0083] The amplified ORF was cloned into the pCR Zero Blunt vector(Invitrogen, Carlsbad, Calif.). The insert was digested by NcoI and XhoIand then subcloned into the isopropyl-1-thio-β-D-galacto-pyranoside(IPTG)-inducible pET19b expression vector (Novagen, Madison, Wis.)designated pPfu-sHSP. The E. coli strain BL21(DE3) (Novagen, Madison,Wis.) carrying pSJS1240 that encodes rare tRNAs for leu and arg in E.coli (Kim et al. 1998d) was used as an expression host. E. coli cellscarrying these 2 plasmids were grown in Luria-Bertani broth containing50 μg/ml ampicillin and spectinomycin to an A₅₉₅ of 0.6.

[0084] The Pfu-sHSP expression was induced by 1 mMisopropyl-1-thio-β-D-galacgtopyranoside (IPTG) for 3 hours. The samestrain carrying pET19b and pSJS1240 served as a control. SDS PAGE of E.coli overexpressing Pfu-sHSP crude extract reveals an additional proteinof 20 kDa in size, which corresponds to the protein molecular weightdeduced from the predicted amino acid sequence. After induction, cellsoverexpressing Pfu-sHSP were harvested and resuspended in 25 mMpotassium phosphate buffer, pH 7.0, 2 mM DTT, 1 mM EDTA (buffer A). Thecells were disrupted using a French Press (SLM instrument, Urbana, Ill.)at 16, 000 psi and centrifuged at 5000×g for 15 minutes.

[0085] Pfu-sHSP appeared in a pellet fraction as indicated by SDS PAGE.The pellet then was dissolved in buffer A by heating at 85° C. for 20minutes. The dissolved pellet was filtered and loaded onto an anionexchange column (MonoQ, Pharmacia Biotech, Uppsala, Sweden) previouslyequilibrated with buffer A. The Pfu-sHSP was eluted using a NaClgradient, at 0.35 M NaCl. The fractions were pooled and concentrated,and the protein appeared homogeneous by SDS PAGE electrophoresisvisualized with silver stain.

[0086] Rabbit polyclonal antibody preparations against the Pfu-sHSP wereobtained by immunization of a rabbit with the purified, recombinantPfu-sHSP (Bio-world, Dublin, Ohio).

[0087] The Pfu-sHSP, containing 167 amino acid residues, wasoverexpressed in E. coli. The SDS PAGE revealed that the molecularweight monomeric Pfu-sHSP was 20 kDa. The amino acid sequence of thisrecombinant protein is shown in FIG. 1 (SEQ ID NO. 2) (GenBank AccessionNo. AF256212).

[0088] The purified Pfu-sHSP was used throughout the experiments.

EXAMPLE 2

[0089] Induction of the Small Heat Shock Protein

[0090]Pyrococcus furiosus was grown as described elsewhere (Gonzalez etal., 1998) at 95° C. for 3 hours and then shifted to 104° C. for 2hours. The cells were collected and frozen immediately and Western blotanalysis was performed (Sambrook et al., 1989) to monitor the Pfu-sHSPinduction.

[0091] The 104° C. temperature is considered a heat shock temperaturefor Pyrococcus furiosus at which it grows optimally at 103° C. Thecontrol culture continued growing at 95° C. without shifting to 104° C.for another 2 hours. A strong signal was observed in the 2-hour heatshocked cells whereas there was no signal observed in the non-heatshocked culture. This indicated that native Pfu-sHSP is heat-inducible,and the protein is not synthesized at sub-inhibitory growthtemperatures.

[0092]FIG. 2 shows the SDS PAGE (15%) and the Western blot analysis ofthe recombinant and the native Pfu-sHSP.

[0093] Specifically, Pyrococcus furiosus was cultured in a modified 20Lfermentor (New Brunswick) or in with S⁰ in the medium without maltose(Adams, M. W. W. 1995, Large-scale growth of hyperthermophiles, p.47-49, In F. T. Robb (ed.) and A. R. Place. Archaea, a laboratorymanual, thermophiles, Cold Spring Harbor Laboratory Press, Plainview,N.Y.). The cultures were incubated at 95° C. for 4 hours, then shiftedto 105° C. for 0, 30, 60, 120 minutes before chilling on ice andharvesting by centrifugation at 7,500×g for 15 minutes. The totalprotein of the cell extracts were measured and an equal amount ofprotein was loaded onto each lane of an SDS gel. Western blot analysiswas conducted. A strong signal at 20 kDa was observed in lanes 2-4,corresponding to 30, 60, and 120 minutes after onset of heat shockwhereas no signal was observed in lane 1 corresponding to the non-heatshocked control culture. This indicated that native Pfu-sHSP wasstrictly heat-inducible, and that protein was not required for rapidgrowth at the optimal growth temperature.

[0094] The expression of mRNA was measured from the gene encoding thePfu-sHSP under heat shock conditions using Northern blot analysis. TotalRNA was isolated from P. furiosus after exposure to 105° C. for 120minutes, compared to the non-heat shocked control culture. Total RNA (4μg) was electrophoresed on a 1.5% agarose gel for Northern blot analysisusing the radiolabelled PCR probe generated by PCR amplification fromprimers Pfu-shspN and Pfu-shspC using ³²P labeled dCTP. Hybridizationwith this probe revealed a transcript of 600 nucleotides correspondingto the size of the putative Pfu-sHSP gene. A radiolabelled probe fromthe gene encoding P. furiosus glutamate dehydrogenase, which isexpressed constitutively, was used as a control.

EXAMPLE 3

[0095] Protection of E. coli Proteins and Cell Viability Under HeatStress

[0096]E. coli strains containing either pPfu-sHSP or pET19b were inducedas described above. The total protection concentration was determinedusing the Bradford protein assay kit (BioRad, Hercules, Calif.). Thetotal proteins in crude extract were diluted in buffer A to aconcentration of 4 mg/ml. The crude extract of each sample was coveredwith mineral oil and heated at 105° C. for 0, 20, 30 and 40 minutes.After cooling down at room temperature, the samples were centrifuged at10,000×g for 5 minutes and the supernatants were collected.

[0097] The soluble proteins were visualized by SDS PAGE (Sambrook etal., 1989).

[0098] The induced cells were also tested for survivability at 50° C.The cultures were shifted rapidly to 50° C. in a water-bath shaker.Samples were taken at 0, 20, 40, 60 and 120 minutes. Samples of theculture were diluted at each time point and plated on Lutria-Bertaniagar containing 50 μg/ml of ampicillin and spectinomycin. Cell viabilitywas determined by counting the colony-forming units after overnightincubation.

[0099] The capacity for Pfu-sHSP to stabilize the full complement ofsoluble proteins in E. coli cell free extracts was determined. All ofthe proteins that can be detected by SDS PAGE in E. coli extractscontaining overexpressed Pfu-sHSP remained soluble after heat treatmentat 105° C., and were recovered in the supernatant fraction aftercentrifugation. The Bradford protein assay indicated that the totalprotein concentration in the supernatant of the control decreasedapproximately 50% whereas those in supernatant with the presence ofPfu-sHSP decreased only 5%.

[0100]FIG. 3 shows the SDS PAGE (15%) analysis of the thermal protectionof E. coli crude extract by Pfu-sHSP at 105° C.

[0101] As the Pfu-sHSP is capable of protecting E. coli proteins fromaggregation in vitro, the issue of whether the Pfu-sHSP could protectcell viability was addressed. The E. coli culture overexpressingPfu-sHSP and a control E. coli culture with pET vector and no insertwere incubated at 50° C. for 2 hours, while viability was measured. Thesingle order death rate of E. coli overexpressing Pfu-sHSP wasapproximately 6-7 fold lower than that of the culture transformed withpET19b and pSJS1240. The difference in viability between protected andunprotected cells after 120 minutes at 50° C. was approximately 3 ordersof magnitude. See Table 1 in respect of the reduction of death rate at50° C. of the E. coli overexpressing Pfu-sHSP in vivo. TABLE 1 Deathrate of E. coli overexpressing Pfu-sHSP E. coli BL21 (DE3) Death rate(log CFU/100 μl/min) PET19b/pSJS1240 0.0058 PPfu-sHSP/pSJS1240 0.038

EXAMPLE 4

[0102] Glutamate Dehydrogenase Activity Assays

[0103] Bovine glutamate dehydrogenase (boGDH) (Sigma, Milwaukee, Wis.)was diluted in EPPS buffer, pH 7.5 to a concentration of 0.9 mg/ml toyield an initial rate of ΔA=0.06/minute. The enzyme was incubated at 56°C. with 2.25 mg/ml of the purified Pfu-sHSP for various times. Thesamples were removed at 0, 2 and 4 minutes and then centrifuged at10,000×g for 2 minutes. The supernatants were tested for the residualenzymatic activity following the method described previously (Robb etal., 1992). The residual activity of boGDH was determined using aBeckman DU640 spectrophotometer fitted with a temperature controller at340 nm and 25° C. The assay mixture contained 100 mM EPPS pH 8.0, 65 mMglutamic acid and 16.25 mM NADP. There was no detectable GDH activity inthe purified Pfu-sHSP.

[0104] The inventors hereof have found that the Pfu-sHSP can protectbovine GDH by aggregation prevention. As a small heat shock protein canprevent aggregation of mixtures of proteins, the inventors hereof testedthe ability of the recombinant Pfu-sHSP to extend the half-life(t_(1/2)) of a pure enzyme in vitro. The mesophilic glutamatedehydrogenase from bovine (boGDH) (Sigma, Milwaukee, Wis.) was used as amodel. The optimal temperature and Tmax of boGDH are 25° C. and 48° C.,respectively. The purified Pfu-sHSP was used to protect boGDH under heattreatment at 56° C.

[0105] The control incubation was without Pfu-sHSP in the same bufferused to store the Pfu-sHSP. In addition, to confirm that the GDHactivity was not from E. coli GDH that may possibly be a minorcontaminant in the purified Pfu-sHSP resulting in increased GDHactivity, the Pfu-sHSP preparation was assayed at high concentration forGDH activity using the same method. No GDH activity was detected in thepurified Pfu-sHSP.

[0106] The t_(1/2) of boGDH incubated with Pfu-sHSP at 56° C. wasincreased approximately four-fold compared to the boGDH control. SeeTable 2 for the results. TABLE 2 Specific activity of boGDH at 56° C.GDH Half-life (seconds) GDH (0.9 mg/ml) 38 GDH/Pfu-sHSP (2.25 mg/ml) 152

[0107] The inventors hereof examined the possibility that the activityof boGDH was protected as a result of prevention of aggregation byPfu-sHSP. The absorbance (A₂₈₀) of the supernatant of boGDH solutionsincubated at 56° C. with or without the presence of Pfu-sHSP wasmeasured. The amount of precipitate had increased and the A₂₈₀ of boGDHwithout Pfu-sHSP was reduced dramatically whereas that of boGDH withPfu-sHSP was not.

[0108] In the experiment where boGDH was incubated alone, the apparentt_(1/2) was approximately 2 minutes whereas the boGDH to which sHSP wasadded did not precipitate at all during the course of the experiment.The activity of the boGDH in the supernatants, on the other hand,declined in both cases, as shown in FIG. 4. Thus, much of the boGDH thatremained in solution was denatured. In this case, the enzyme wasmaintained in solution but not preserved from denaturation. This is animportant result that indicates that the probable mode of action ofPfu-sHSP is preventing non-specific aggregation of proteins therebyallowing them to be recruited to either refolding or protein turnoverpathways.

EXAMPLE 5

[0109] DNA Polymerase Protection by Recombinant Protein

[0110] 1.25 Taq Polymerase (Sigma, catalog number D1806) was incubatedwith and without 7.5 μg of Pfu-sHSP in 40 μl of Taq polymerase bufferwith 15 mM MgCl₂ (Sigma) at −10 and 50° C. for 16 hours. PCR was doneusing the incubated Taq polymerase. The PCR reaction was carried out asfollows:

[0111] 1. 1× Taq polymerase buffer

[0112] 2. 1 μM forward and reverse primers

[0113] 3. 0.2 mM dNTP

[0114] 4. 1.25U Taq polymerase

[0115] The PCR cycles were carried out according to the following chartof cycles: Cycles 1 cycle 28 cycles 1 cycle Temperature 94° C. 94° C.52° C. 72° C. 52° C. 72° C. Time 2 minutes 30 seconds 30 seconds 30seconds 2 minutes 2 minutes

[0116] The PCR products were visualized in 1% agarose gelelectrophoresis.

[0117] Taq polymerase was incubated with sHSP at 50° C. for 20 hours.PCR of a target containing the gene encoding sHSP was performed usingPfu-sHSP primers and Pfu genomic DNA.

[0118] The results of the agarose gel electrophoresis showed thefollowing: Lane 1 100 bp ladder (Promega) Lane 2 PCR with normal Taqpolymerase Lane 3 PCR with normal Taq polymerase and 1 μl of sHSP (finalconcentration) Lane 4, 5 PCR with Taq incubated at 50° C. 20 hours with1 μl of dH₂O Lane 6, 7 PCR with Taq incubated with sHSP at 50° C. 20hours

[0119]FIG. 5 shows the results of the agarose gel electrophoresis.

EXAMPLE 6

[0120] Limitation of the Polymerase Molecules to Carry Out PCR Products

[0121] This procedure is carried out by the following steps.

[0122] 1. Taq polymerase is diluted into several dilutions.

[0123] 2. The diluted Taq polymerase is incubated with and without smallheat shock protein.

[0124] 3. The PCR process is performed.

[0125] 4. A comparison is made of the PCR products from Taq polymeraseincubated with and without the sHSP to establish the lowest dilutionthat carries out the PCR products.

EXAMPLE 7

[0126] Limitation of the Polymerase Molecule to Sequence PCR Product

[0127] This procedure is carried out by the following steps.

[0128] 1. Thermosequenase used for sequencing PCR products is dilutedinto several dilutions.

[0129] 2. The enzyme from step 1 is incubated with and without sHSP.

[0130] 3. The PCR process is performed.

[0131] 4. An automated DNA sequencer is employed to carry out thesequencing.

[0132] 5. The sequencing peaks as a result from the enzyme incubatedwith and without sHSP are compared.

EXAMPLE 8

[0133] Effect of Pfu-sHSP on Taq Polymerase Concentration in PCRReaction

[0134] The effect of Pfu-sHSP on concentration of Taq polymerase (FisherScientific) in a PCR reaction medium was assessed in three separateexperiments, whose results are shown in the electrophoretic gelsdepicted in FIGS. 6-8.

[0135]FIG. 6 depicts an electrophoretic gel (1% agarose) showing theeffect of Pfu-sHSP on Taq polymerase concentration in PCR reaction, atan sHSP concentration of 0.2 μg/μl. The respective lanes 1-7 shown inthe figure are as follows: Lane 1 100 bp marker Lane 2  0.025 U/μl +sHSP Lane 3  0.025 U/μl − sHSP Lane 4  0.005 U/μl + sHSP Lane 5  0.005U/μl − sHSP Lane 6 0.0025 U/μl + sHSP Lane 7 0.0025 U/μl − sHSP

[0136]FIG. 7 depicts another electrophoretic gel (1% agarose) showingthe effect of Pfu-sHSP on Taq polymerase concentration in PCR reaction,at an sHSP concentration of 0.2 μg/μl. The respective lanes 1-7 shown inthe figure are as follows: Lane 1 100 bp marker Lane 2  0.025 U/μl +sHSP Lane 3  0.025 U/μl − sHSP Lane 4  0.005 U/μl + sHSP Lane 5  0.005U/μl − sHSP Lane 6 0.0025 U/μl + sHSP Lane 7 0.0025 U/μl − sHSP

[0137]FIG. 8 depicts yet another electrophoretic gel (1% agarose)showing the effect of Pfu-sHSP on Taq polymerase concentration in PCRreaction, at an sHSP concentration of 0.2 μg/μl. The respective lanes1-7 shown in the figure are as follows: Lane 1 100 bp marker Lane 2 0.025 U/μl + sHSP Lane 3  0.025 U/μl − sHSP Lane 4  0.005 U/μl + sHSPLane 5  0.005 U/μl − sHSP Lane 6 0.0025 U/μl + sHSP Lane 7 0.0025 U/μl −sHSP

[0138] The results are consistent in the respective experimental runs,and show that the presence of Pfu-sHSP in the PCR reaction volume wasassociated with a higher concentration of Taq polymerase in the reactionmedium than in the corresponding reaction volumes in which the Pfu-sHSPheat shock protein was not present.

[0139] The results thereby demonstrate the advantage of utilizing a heatshock protein in accordance with the invention, in PCR operations. Thepresent invention contemplates a PCR kit including a heat shock proteincomponent for use in the PCR reaction medium to resist aggregation andprecipitation of the polymerase component of the reaction mixture. ThePCR kit in a specific embodiment may therefore include PCR primers, Taqpolymerase, deoxyribonucleoside triphosphates and an HSP. Alternatively,the PCR kit may include an HSP together with at least one other of thecomponents specified in the preceding sentence.

EXAMPLE 9

[0140] Cloning, Overexpression and Purification of the RecombinantPfu-tsHSP Protein

[0141] The region encoding the Pfu-tsHSP gene (SEQ ID NO. 5) (FIG. 9)was amplified from Pryrococcus furiosus genomic DNA by PCR using twoprimers: Pfu-tsHSPN with an NcoI site (underlined)(5′GCCATGGTGAGGAGAATAAGAAGATGG) (SEQ ID NO. 3) and Pfu-tsHSPC with anXhoI site (underlined) (5′ACTCGAGCTACTTTGTTGGGTGCTTCTTTGGAACTCTGATC)(SEQ ID NO. 7).

[0142] The amplified ORF was cloned into the pCR Zero Blunt vector(Invitrogen, Carlsbad, Calif.). The insert was digested by NcoI and XhoIand then subcloned into the isopropyl-1-thio-β-D-galacto-pyranoside(IPTG)-inducible pET19b expression vector (Novagen, Madison, Wis.)designated pPfu-shsp. The E. coli strain BL21(DE3) (Novagen, Madison,Wis.) carrying pSJS1240 that encodes rare tRNAs for leu and arg in E.coli (Kim et al. 1998d) was used as an expression host. E. coli cellscarrying these 2 plasmids were grown in Luria-Bertani broth containing50 μg/ml ampicillin and spectinomycin to an A₅₉₅ of 0.6.

[0143] The Pfu-tsHSP expression was induced by 1 mMisopropyl-1-thio-β-D-galacgtopyranoside (IPTG) for 3 hours. The samestrain carrying pET19b and pSJS1240 served as a control. SDS PAGE of E.coli overexpressing Pfu-tsHSP crude extract reveals an additionalprotein of 19 kDa in size, which corresponds to the protein molecularweight deduced from the predicted amino acid sequence. After induction,cells overexpressing Pfu-tsHSP were harvested and resuspended in 25 mMpotassium phosphate buffer, pH 7.0, 2 mM DTT, 1 mM EDTA (buffer A). Thecells were disrupted using a French Press (SLM instrument, Urbana, Ill.)at 16,000 psi and centrifuged at 5000×g for 15 minutes.

[0144] Pfu-tsHSP appeared in a pellet fraction as indicated by SDS PAGE.The pellet then was dissolved in buffer A by heating at 85° C. for 20minutes. The dissolved pellet was filtered and loaded onto an anionexchange column (MonoQ, Pharmacia Biotech, Uppsala, Sweden) previouslyequilibrated with buffer A. The Pfu-tsHSP was eluted using an NaClgradient, at 0.35 M NaCl. The fractions were pooled and concentrated,and the protein appeared homogeneous by SDS PAGE electrophoresisvisualized with silver stain.

[0145] Rabbit polyclonal antibody preparations against the Pfu-tsHSPwere obtained by immunization of a rabbit with the purified, recombinantPfu-tsHSP (Bio-world, Dublin, Ohio).

[0146] The Pfu-tsHSP, containing 156 amino acid residues, wasoverexpressed in E. coli. The SDS PAGE revealed that the molecularweight monomeric Pfu-tsHSP was 19 kDa (sHSP19). The amino acid sequenceof this recombinant protein is shown in FIG. 9 (SEQ ID NO. 6).

[0147] The purified Pfu-tsHSP was used in the following experiment.

EXAMPLE 10

[0148] Comparison of Native Pfu-sHSP and Recombinant Pfu-tsHSP UnderHeat Stress

[0149]E. coli strains containing recombinant pPfu-tsHSP, nativepPfu-sHSP and a control pET19b were induced as described above. Thetotal protection concentration was determined using the Bradford proteinassay kit (BioRad, Hercules, Calif.). The total proteins in cell freeextracts were heated at 105° C. for 0, 10, 20 and 40 minutes. Aftercooling down at room temperature, the samples were centrifuged at10,000×g for 5 minutes and the supernatants were collected.

[0150] The soluble proteins were visualized by SDS PAGE (Sambrook etal., 1989). FIG. 10 shows the SDS PAGE (15%) analysis of the thermalprotection of E. coli cell extract by Pfu-tsHSP at 105° C. Specifically,polyacrylamide gel resolution of the soluble proteins in a cell-freeextract of E. coli shows protection of the cell free extract by nativesHSP chaperone in lanes 2, 5 8 and 11 which were heated to 105° C. for0, 10, 20 and 40 minutes, respectively. The recombinant Pfu-tsHSP, foundin lanes 3, 6, 9 and 12 shows identical protein stabilization as that ofthe native type. The truncated Pfu-tsHSP small heat protein, lacking 11amino acids from the carboxy end of the native sHSP, is unable to forminter-dimer crosslinks and as such has a fixed molecular weight of 19KDa.

[0151] The foregoing results evidence the utility of the inventivemethod for stabilizing a protein solution including a first protein,against heat-mediated agglomeration of such first protein in thesolution, by incorporating in the solution a heat shock protein that isnon-endogenous with respect to the first protein.

[0152] The clear effects of Pfu-sHSP and/or Pfu-tsHSP on proteinstabilization and the increased thermotolerance it confers on E. colireflect the mechanisms of its action in vivo. Protein stabilization isconsistent with the maintenance of solubility of proteins, therebypromoting refolding and assembly. It is highly unexpected andfundamentally surprising that a component of the adaptive response of anarchaeum growing at 100° C., such as P. furiosus, can enhance the heatresistance of organisms (e.g., E. coli) growing at much lowertemperatures. The invention therefore embodies a substantial advance inthe art of heat shock proteins, implicating a wide variety ofapplications in which such heat shock proteins confer enhancedthermotolerance, survivability and utility in the context ofsupraordinary thermal exposure conditions.

[0153] The disclosures of all references cited herein are herebyincorporated herein in their respective entireties.

[0154] While the invention has been described herein with reference tovarious illustrative features, aspects, and embodiments, it will beappreciated that the utility of the invention is not thus limited, butrather extends to and encompasses other variations, modifications andother embodiments, as will readily suggest themselves to those ofordinary skill in the art. Accordingly, the invention is to be broadlyinterpreted and construed as including such other variations,modifications and other embodiments, within the spririt and scope of theinvention as hereinafter claimed.

1 7 1 712 DNA Pyrococcus furiosus 1 tcttttttgg agtatttttg attgttcggtaaattctact cttatcgaaa atatttataa 60 accccaaata atttaataac taatggtaaccaaaagtggg agggggtgag agagatggtg 120 aggagaataa gaagatggga catatgggatccattcgacc taataaggga aatacaagag 180 gaaattgatg caatgttcga tgaattcttcagcaggccaa ggctctggac ttacagaagg 240 tggagcgagc cagcaatgta tgaggagagagtaggagaag tctggagaga gccattcgtt 300 gatatctttg acaacggaga tgagtttgtaatcacggcag agcttccagg agtgagaaaa 360 gaagacatca aagtgagggt tacagaggatacagtataca ttgaggccac agttaagagg 420 gagaaagaat tagaaagaga aggagcagtgagaatagaga gatactttac agggtataga 480 agagccatca ggcttccaga agaagttattccagagaagg caaaggccaa gtacaacaac 540 ggagtgcttg agatcagagt tccaaagaagcacccaacaa agaaggagag tgaaggattc 600 gaagttaaag ttgaatagct ttagtacccttctttcttga ttatttggaa atatttttgg 660 aggtattggt tctattatca attaattccttttattttaa aatccttgga tc 712 2 167 PRT Pyrococcus furiosus 2 Met Val ArgArg Ile Arg Arg Trp Asp Ile Trp Asp Pro Phe Asp Leu 1 5 10 15 Ile ArgGlu Ile Gln Glu Glu Ile Asp Ala Met Phe Asp Glu Phe Phe 20 25 30 Ser ArgPro Arg Leu Trp Thr Tyr Arg Arg Trp Ser Glu Pro Ala Met 35 40 45 Tyr GluGlu Arg Val Gly Glu Val Trp Arg Glu Pro Phe Val Asp Ile 50 55 60 Phe AspAsn Gly Asp Glu Phe Val Ile Thr Ala Glu Leu Pro Gly Val 65 70 75 80 ArgLys Glu Asp Ile Lys Val Arg Val Thr Glu Asp Thr Val Tyr Ile 85 90 95 GluAla Thr Val Lys Arg Glu Lys Glu Leu Glu Arg Glu Gly Ala Val 100 105 110Arg Ile Glu Arg Tyr Phe Thr Gly Tyr Arg Arg Ala Ile Arg Leu Pro 115 120125 Glu Glu Val Ile Pro Glu Lys Ala Lys Ala Lys Tyr Asn Asn Gly Val 130135 140 Leu Glu Ile Arg Val Pro Lys Lys His Pro Thr Lys Lys Glu Ser Glu145 150 155 160 Gly Phe Glu Val Lys Val Glu 165 3 27 DNA ArtificialSequence Synthetic Construct 3 gccatggtga ggagaataag aagatgg 27 4 34 DNAArtificial Sequence Synthetic Construct 4 actcgagcta ttcaactttaacttcgaatc cttc 34 5 468 DNA Pyrococcus furiosus 5 atggtgagga gaataagaagatgggacata tgggatccat tcgacctaat aagggaaata 60 caagaggaaa ttgatgcaatgttcgatgaa ttcttcagca ggccaaggct ctggacttac 120 agaaggtgga gcgagccagcaatgtatgag gagagagtag gagaagtctg gagagagcca 180 ttcgttgata tctttgacaacggagatgag tttgtaatca cggcagagct tccaggagtg 240 agaaaagaag acatcaaagtgagggttaca gaggatacag tatacattga ggccacagtt 300 aagagggaga aagaattagaaagagaagga gcagtgagaa tagagagata ctttacaggg 360 tatagaagag ccatcaggcttccagaagaa gttattccag agaaggcaaa ggccaagtac 420 aacaacggag tgcttgagatcagagttcca aagaagcacc caacaaag 468 6 156 PRT Pyrococcus furiosus 6 MetVal Arg Arg Ile Arg Arg Trp Asp Ile Trp Asp Pro Phe Asp Leu 1 5 10 15Ile Arg Glu Ile Gln Glu Glu Ile Asp Ala Met Phe Asp Glu Phe Phe 20 25 30Ser Arg Pro Arg Leu Trp Thr Tyr Arg Arg Trp Ser Glu Pro Ala Met 35 40 45Tyr Glu Glu Arg Val Gly Glu Val Trp Arg Glu Pro Phe Val Asp Ile 50 55 60Phe Asp Asn Gly Asp Glu Phe Val Ile Thr Ala Glu Leu Pro Gly Val 65 70 7580 Arg Lys Glu Asp Ile Lys Val Arg Val Thr Glu Asp Thr Val Tyr Ile 85 9095 Glu Ala Thr Val Lys Arg Glu Lys Glu Leu Glu Arg Glu Gly Ala Val 100105 110 Arg Ile Glu Arg Tyr Phe Thr Gly Tyr Arg Arg Ala Ile Arg Leu Pro115 120 125 Glu Glu Val Ile Pro Glu Lys Ala Lys Ala Lys Tyr Asn Asn GlyVal 130 135 140 Leu Glu Ile Arg Val Pro Lys Lys His Pro Thr Lys 145 150155 7 41 DNA Artificial Sequence Synthetic Construct 7 actcgagctactttgttggg tgcttctttg gaactctgat c 41

1. A purified and isolated nucleic acid sequence encoding a heat shockprotein comprising SEQ ID NO.
 1. 2. A recombinant DNA vector forexpression of Pfu-sHSP, wherein the recombinant DNA vector comprises SEQID NO. 1 of claim
 1. 3. A transformed cell capable of expressingPfu-sHSP, wherein the transformed cell comprises a recombinant DNAvector comprising SEQ ID NO. 1 of claim
 2. 4. An isolated proteinencoded by the nucleic acid comprising SEQ ID NO.
 5. 5. An isolatedprotein comprising the amino acid sequence of SEQ ID NO.
 6. 6. Acomposition comprising the protein of claim
 4. 7. A compositioncomprising the protein of claim
 5. 8. A method of protecting andextending the durability of a recombinant DNA polymerase, comprising thesteps of: (a) adding a heat shock protein comprising the amino acidsequence of SEQ. ID NO. 6 according to claim 5 to a buffer solutioncontaining said polymerase; (b) incubating the solution at elevatedtemperature for a predetermined time; (c) adding components necessaryfor polymerase chain reaction; (d) thermocycling said reaction toproduce product from amplification of genomic deoxyribonucleic acid; and(e) examining said product by gel electrophoresis.
 9. A method ofmaintaining proteins in solution, comprising the steps of: adding a heatshock protein comprising the amino acid sequence of SEQ ID NO. 6according to claim 5 to the solution; elevating the temperature of thesolution; and measuring the enzymatic activity by absorbance.
 10. A PCRkit, comprising a protein as in claim 4 and at least one other PCRreagent.
 11. A PCR kit, comprising a protein as in claim 5 and at leastone other PCR reagent.
 12. A method of enhancing the stability of Taqpolymerase in a PCR operation, comprising conducting said PCR operationin the presence of a heat shock protein (HSP) comprising the amino acidsequence of SEQ ID NO. 6 according to claim
 5. 14. The method of claim13, wherein said HSP comprises Pfu-tsHSP.
 15. The method of claim 13,wherein said HSP comprises a protein encoded by the nucleic acidcomprising SEQ ID NO.
 5. 16. A composition comprising (i) a biologicalcomponent and (ii) a heat shock protein (HSP) comprising the amino acidsequence of SEQ ID NO.
 6. 17. The composition of claim 16, wherein saidbiological component comprises a material selected from the groupconsisting of biological cells, and fractions and components thereof.18. The composition of claim 16, wherein said biological componentcomprises an enzyme.
 19. The composition of claim 16, wherein saidbiological component comprises a microbial culture.
 20. The compositionof claim 16, comprising a solution.
 21. The composition of claim 20,wherein the biological component comprises a protein, and the heat shockprotein comprising the amino acid sequence of SEQ ID NO. 6 is present inan effective amount to enhance resistance of the protein to aggregationand/or precipitation upon heat exposure of the composition.
 22. Thecomposition of claim 16, wherein said heat shock protein comprises aPfu-tsHSP.
 23. The composition of claim 16, wherein said heat shockprotein comprises a protein encoded by the nucleic acid comprising SEQID NO.
 5. 24. A PCR kit including PCR primers, Taq polymerase,deoxyribonucleoside triphosphates and a heat shock protein (HSP)comprising the amino acid sequence of SEQ ID NO.
 6. 25. The PCR kit ofclaim 24, wherein said HSP comprises a Pfu-tsHSP.
 26. The PCR kit ofclaim 24, wherein said HSP comprises a protein encoded by the nucleicacid comprising SEQ ID NO.
 5. 27. A method of stabilizing a proteinsolution, including a first protein therein, against heat-mediatedagglomeration of said first protein in the solution, comprisingincorporating in the solution a heat shock protein that isnon-endogenous with respect to said first protein, wherein said heatshock protein comprises the amino acid sequence of SEQ ID NO. 6according to claim
 5. 28. The method of claim 27, wherein said heatshock protein comprises a Pfu-tsHSP.
 29. A PCR kit, comprising a sHSP19heat shock protein, and optionally including at least one componentselected from the group consisting of (i) PCR primers, (ii) a polymeraseeffective for PCR and (iii) deoxyribonucleoside triphosphates.
 30. Amethod of amplifying a nucleic acid sequence by a PCR reaction,comprising conducting said PCR reaction in the presence of sHSP19 heatshock protein.
 31. A purified and isolated nucleic acid sequenceencoding a heat shock protein comprising SEQ ID NO.
 5. 32. A recombinantDNA vector for expression of Pfu-tsHSP, wherein the recombinant DNAvector comprises SEQ ID NO. 5 according to claim
 31. 33. A transformedcell capable of expressing Pfu-tsHSP, wherein the transformed cellcomprises a recombinant DNA vector comprising SEQ ID NO. 5 according toclaim 32.